Glioblastoma multiforme (GBM) is the most common and deadly form of brain cancer found in humans. The median time to tumor recurrence after resection is just 6.9 months after common treatment; the median survival time from diagnosis is just 1-2 years. Its uncommonly aggressive migration away from the host tumor is likely responsible for the inability to completely surgically resect the tumor, rendering a curative treatment currently out of reach. The broad goals of this project are to develop more in depth understanding of GBM migration away from the host tumor. It is widely recognized that GBM dissemination occurs along defined tracks in the brain, including vasculature and white matter tracts, but this phenomenon is poorly understood. The spatial inhomogeneities presented by these tracks - including, stiffness and ligand inhogenieties - may help to guide cells away from the host tumor. In this proposal, we seek to create an in vitro model system to replicate the spatial inhomogoneities present in tumors and ask whether spatial stiffness and ligand inhomogeneities can enhance GBM cell migration. To accomplish this, this proposal has three specific aims. Aim 1 is to develop a three-dimensional hyaluronic acid platform with orthogonal control of matrix stiffness and ligand attachment. We will use two- photon activation of orthogonal chemistries to achieve this goal, which will allow us to recreate the tumor environment in vitro. Using this substrate, we will investigate aim 2: to test the hypothesis that stiffness inhomogeneities and local ligand type promote the dissemination of glioblastoma cells. Geometries designed to mimic blood vessels will be created such that their stiffness and ligand density and type are varied systematically to determine what conditions contribute to most efficient cell migration. Our last aim is to investigate the intracellular signaling pathways by which cells may interpret these cues. Aim 3 is to investigate the role of focal adhesion kinase in cell migration through peptide activated three-dimensional hyaluronic acid matrices with stiffness inhomogeneities. Importantly, FAK is often deregulated in many cancers and gliomas, suggesting it may play an important role in tumor dissemination. We expect this proposal to significantly contribute to the understanding of glioblastoma cell migration, specifically elucidating the factors that may promote dissemination from the host tumor. The results of this study will not only increase our understanding of the disease, but may also suggest possible therapeutic targets to inhibit cell migration.